Atopic dermatitis (AD) is a pruritic chronic inflammatory skin disease associated with significant morbidity and occupational disability affecting 17% of children and nearly 2% of adults in the U.S. Studies have highlighted the importance of S. aureus infection, colonization and toxin production in AD severity/exacerbation. The overall goal of this competing renewal R01 grant application (5 R01 AR41256-18) will be to determine how colonization with toxin producing S. aureus in AD patients alters inflammatory response in the skin. As well, we wish to investigate novel mechanisms by which Th2 environment in combination with toxin production by S. aureus enhance skin inflammation. Our previous studies of staphylococcal superantigens primarily focused on mononuclear cell activation by these toxins in the skin. New preliminary data show that staphylococcal superantigens (staphylococcal enterotoxin B, SEB) also exert effects directly on keratinocytes and contribute to the skin inflammation by inducing proinflammatory and Th2 cell skin homing chemokines. Global gene expression profiling of SEB stimulated human keratinocytes demonstrates protease activation by SEB and suggests that SEB may enhance allergic skin sensitization due to changes in extracellular matrix that can affect allergen absorption and inflammation, which we will explore in this proposal. We present evidence for Th2 cytokine potentiation of keratinocyte responses to SEB and propose that this occurs either through synergism with SEB induced pathways and/or increased expression of a specific receptor to SEB, therefore amplifying inflammatory responses in atopic skin. Finally, we demonstrate that staphylococcal pore forming alpha-toxin, that often coincides with superantigen production by S. aureus, can potentiate responses to staphylococcal products by facilitating their intracellular entry via toxin-induced pores, thereby allowing the engagement of intracellular receptors. Th2 cytokines may increase alpha-toxin binding to keratinocytes, affecting pore formation and intensifying keratinocyte responses to staphylococcal products. The specific aims of this proposal will be: first, to determine the effects of staphylococcal superantigens on inflammatory responses of keratinocytes, focusing on gene and protein expression profiling in superantigen activated human keratinocytes and skin samples from AD patients colonized with superantigen producing S. aureus, in vivo inflammatory responses and allergen absorption in murine skin following epicutaneous application of SEB; second, to explore the modulatory role of Th2 cytokines in responses to staphylococcal superantigens in the skin by analyzing changes in gene and protein expression in keratinocytes, assessing inflammatory responses to SEB in STAT6 transgenic mice, exploring signaling pathways engaged by SEB and Th2 cytokines, and defining the specific SEB receptor; finally, to investigate the immunomodulatory role of staphylococcal alpha-toxin in keratinocyte responses to SEB/other staphylococcal products. Studies will likely identify therapeutic approaches in the control of skin inflammation initiated by staphylococcal toxins and atopy.